Kaposi's sarcoma(KS)-associated herpesvirus(KSHV/HHV8) is a DNA tumor virus etiologically associated with KS and several other malignancies. The applicant's long-range goal is to use KSHV as a paradigm to understand the molecular mechanism of virus-induced pathogenesis, providing biologic basis for preventative and therapeutic purposes. The overall hypothesis is that KSHV encodes specific genes to evade host antiviral defenses and complete viral life cycle. An array of KSHV genes has been shown to deregulate cellular functions when cloned and examined individually. However, their precise functions in viral infection and KSHV-related pathogenesis remain unclear. The lack of an efficient infection model has hampered the investigation of KSHV infection, replication, and genetic manipulation so far. We have recently cloned the entire KSHV genome into a bacteria artificial chromosome (BAC) and reconstituted it in 293 cells. Recombinant virions can infect 293 cells with approximately 100% primary infection efficiency, and establish stable latent infection. Recombinant genomes can also be easily recovered in bacteria. The objective of this application is to develop cellular models for KSHV infection, replication, cellular transformation, and genetic analysis, and to use it to examine functions of viral immediate-early (IE) genes. The central hypothesis is that recombinant KSHV infection of 293 cells and human primary endothelial cells closely mimics the natural viral life cycle, and could be used as cellular models for viral infection and replication. The rationale is that such cellular models are vital for understanding KSHV virology and KSHV-related pathogenesis, and IE genes have critical roles in viral infection and replication. There are three specific aims: 1) To determine the expression patterns of viral and cellular genes, and phenotypic characteristics of recombinant KSHV infection and replication in 293 cells; 2) To infect and transform human primary endothelial cells with recombinant virions, establish stable cell lines, and determine their virology and cellular biology; 3) To define the functions of KSHV IE genes in viral infection, replication, and cellular transformation by examining viral mutants using the cellular models. The proposed work is innovative, because currently there is no efficient cellular model for KSHV infection and genetic manipulation. It is our expectation that these studies will define the virology and cellular biology of KSHV primary infection, latent and lytic replication, cellular transformation and functions of IE genes. These studies will be significant, because they will establish cellular models for examining KSHV infection and functional dissections of individual viral genes, which could provide insight into the pathogenesis of KSHV-related malignancies. The cellular models will also be useful for the development of anti-KSHV vaccines and therapeutic drugs.